Backbone Dynamics of TEM-1 Determined by NMR: Evidence for a Highly Ordered Protein

Abstract

Backbone dynamics of TEM-1 β-lactamase (263 amino acids, 28.9 kDa) were studied by ¹⁵N nuclear magnetic resonance relaxation at 11.7, 14.1, and 18.8 T. The high quality of the spectra allowed us to measure the longitudinal relaxation rate (R₁), the transverse relaxation rate (R₂), and the {¹H}−¹⁵N NOE for up to 227 of the 250 potentially observable backbone amide groups. The model-free formalism was used to determine internal motional parameters using an axially anisotropic model. TEM-1 exhibits a small prolate axial anisotropy (D_∥/D_⊥ = 1.23 ± 0.01) and a global correlation time (τ_m) of 12.41 ± 0.01 ns. The unusually high average generalized order parameter (S²) of 0.90 ± 0.02 indicates that TEM-1 is one of the most ordered proteins studied by liquid-state NMR to date. Although the Ω-loop has a high degree of order in the picosecond-to-nanosecond time scale (mean S² value of 0.90 ± 0.02), we observed the presence of microsecond-to-millisecond time scale motions for this loop, as for the vicinity of the active site. These motions could be relevant for the catalytic function of TEM-1. Amide exchange experiments were also performed, and several amide groups were not exchanged after 12 days, an indication that global motions in TEM-1 are also very limited. Although detailed dynamics characterization by NMR cannot be readily applied to TEM-1 in the presence of relevant substrates, the unusual picosecond-to-nanosecond dynamics behavior of TEM-1 presented here will be essential to the validation and improvement of future molecular dynamics simulations of TEM-1 in the presence of functionally relevant substrates.